Restoration of epithelial tissue after tissue injury is a complex process, which includes several critical events, including deposition of extracellular matrix ("ECM"), tissue remodeling, and angiogenesis. These events are coordinated with epithelial cell migration and proliferation to restore the epithelial and/or mucosal barrier (i.e., in epithelial tissues such as tracheal epithelium which secrete mucous). The coordination of these events is believed to involve the interaction between different classes of cells as well as between cells and their extracellular matrix.
Failure of re-epithelialization after injury has been observed in the cornea (Fini et al., "Expression of Collagenolytic/Gelatinolytic Metalloproteinases by Normal Cornea," Invest. Opthalmol. Vis. Sci. 31:1779-1788 (1990)) and in chronic wounds (Stacey et al., "Tissue and Urokinase Plasminogen Activators in the Environs of Venous and Ischemic Leg Ulcers," Br. J. Surg. 80:595-599 (1993); Wysocki et al., "Wound Fluid from Chronic Leg Ulcers Contains Elevated Levels of Metalloproteinases MMP-2 and MMP-9," J. Invest. Dermatol. 101:64-68 (1993); Madlener et al., "Matrix Metalloproteinases (MMPs) and their Physiological Inhibitors (THAPs) are Differentially Expressed During Excisional Skin Wound Repair," Exp. Cell Res. 242:201-210 (1998); Di Colandrea et al., "Epidermal Expression of Collagenase Delays Wound-healing in Transgenic Mice," J. Invest. Dermatol. 11:1029-1033 (1998)). Proteinases that destroy the basement membrane over which epithelial cells migrate have been implicated as mediators in impaired capacity to re-epithelialize.
Tissue remodeling during wound healing is critical for repair as cellular migration over an appropriate ECM requires controlled and tightly regulated proteolytic degradation of the ECM, with consequent activation or release of matrix-bound growth factors (Clark, "Basics of Cutaneous Wound Repair," J. Dermatol. Surg. Oncol. 19:693-706 (1993); Salo et al., "Expression of Matrix Metalloproteinase-2 and -9 During Early Human Wound Healing," Lab. Invest. 70:176-182 (1994); Vaalamo et al., "Patterns of Matrix Metalloproteinase and TIMP-1 Expression in Chronic and Normally Healing Human Cutaneous Wounds," Br. J. Dermatol. 135:5259-59(1996); Moses et al., "Temporal Study of the Activity of Matrix Metalloproteinases and Their Endogenous Inhibitors During Wound Healing," J. Cell. Biochem. 60:379-386 (1996); Martin "Wound Healing--Aiming for Perfect Skin Regeneration," Science 276:75-81 (1997); Arumagam et al., "Temporal Activity of Plasminogen Activators and Matrix Metalloproteinases During Cutaneous Wound Repair," Surgery 125:5887-593 (1999)).
As with the above-described tissues, re-epithelialization of injured tracheal tissues is often incomplete. The ability of respiratory epithelial cells ("RECs") to migrate and proliferate and restore denuded areas of the large conducting airway after injury is poor. Post-trauma restoration is pathologically manifested by the exuberant proliferation of granulation tissue and replacement of the normal respiratory epithelium with fibroblasts (Clark, "The Commonality of Cutaneous Wound Repair and Lung Injury," Chest. 99(Suppl.):57S-60S (1991); Grillo, "Tracheal Replacement," Ann. Thorac. Surg. 49:864-865 (1990)). This often leads to scar formation, airway stenosis, and eventual physiologic compromise of the host respiratory tract.
There is currently no effective way to study events of re-epithelialization after injury, particularly with respect to the intraluminal events surrounding tracheal repair. Present approaches to tracheal repair include resection and reanastomosing the injured airway, replacement of the damaged portion by synthetic material, and use of autologous tissue for reconstruction of the tracheal defect (Letang et al., "Experimental Reconstruction of the Canine Trachea with a Free Revascularized Small Bowel Graft," Ann. Thorac. Surg. 49:955-958 (1990); Mulliken et al., Abstract, "The Limits of Tracheal Resection with Primary Anastomosis: Further Anatomical Studies in Man," J. Thorac. Cardiovasc. Surg. 55:418 (1968); Neville et al., "Prosthetic Reconstruction of the Trachea and Carina," J. Thorac. Cardiovasc. Surg. 72:525-536 (1976)). Recently, tissue engineering approaches have been taken, including forming an in vivo tracheal cartilaginous scaffolding by injecting dissociated chondrocytes into a preformed synthetic construct (Hirano et al., "Hydroxylapatite for Laryngotracheal Framework Construction. Ann. Otol. Rhinol. Laryngol. 98:713-717 (1989); Okumura et al., "Experimental Study of a New Tracheal Prosthesis Made from Collagen Grafted Mesh," Trans. Am. Soc. Artif. Organs. 37:M317-M319 (1991); Langer et al., "Tissue Engineering," Science 260:920-926 (1993)). Such devices were of limited success owing to lack of reepithelialization. In the case of synthetic replacement, migration of the prosthesis can occur and may result in chronic ulceration, and even fatal hemorrhage (Grillo, "Tracheal Replacement", Ann. Thorac. Surg. 49:864-865 (1990)).
A frequent problem seen in tracheal repair with synthetic or autologous materials is the failure of luminal surface reepithelialization. Failure of reepithelialization to reestablish luminal integrity is an important reason why no acceptable surgical procedure exists for the repair of extended segments of trachea compromised by inhalation injury, congenital anomalies, or neoplastic disease.
Why the rate of reepithelialization in the large conducting airway is different from that seen within other epithelial-lined or -covered surfaces is unclear. The phenomenon of "slowed" reepithelialization is seen after both ablative surgical reconstruction and denudation injury, where the epithelium and basement membrane are removed with an intact cartilaginous superstructure (e.g., inhalation injury).
One of the difficulties in understanding the relationship between epithelium and its underlying substructure (cartilage and submucosa) is the inaccessibility of the tissue for direct observation. It would be desirable, therefore, to provide an in vitro cell culture which includes a developed substructure or cartilaginous layer which can be used to study epithelial cell development.
The present invention is directed to overcoming these and other deficiencies in the art.